DOCSLIB.ORG

Allosteric Network in Ube2t Drives Specificity for RING E3 Catalysed

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Allosteric Network in Ube2t Drives Specificity for RING E3 Catalysed bioRxiv preprint doi: https://doi.org/10.1101/429076; this version posted September 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Viduth K Chaugule1, 2*, Connor Arkinson1, 2, Rachel Toth2 and Helen Walden1, 2* 2 1Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life 3 Sciences, University of Glasgow, Glasgow, UK 4 2MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences 5 University of Dundee, Dundee, UK 6 * Corresponding authors. E-mail: [email protected], 7 [email protected] 8 9 Running title: E2 allostery for specific ubiquitination 1 bioRxiv preprint doi: https://doi.org/10.1101/429076; this version posted September 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 10 Abstract 11 In eukaryotes, DNA damage repair is implemented by a host of proteins that are coordinated 12 by defined molecular signals. One such signal that transpires during the Fanconi Anemia 13 (FA) - interstrand crosslink (ICL) repair pathway is the site-specific monoubiquitination of 14 FANCD2 and FANCI proteins by a large, multi-protein FA core complex. The mechanics for 15 this exquisitely specific monoubiquitin signal has been elusive. Here we show FANCL, the 16 RING E3 module of the FA core complex, allosterically activates its cognate E2 Ube2T for 17 monoubiquitination by a mechanism distinct from the typical RING-based catalysis. FANCL 18 triggers intricate re-wiring of Ube2T’s intra-residue network thus activating the E2 for 19 precision targeting. This network is intrinsically regulated by conserved gates and loops 20 which can be engineered to yield Ube2T variants that enhance FANCD2 ubiquitination by 21 ~30-fold without compromising on target specificity. Finally, we also uncover allosteric 22 networks in other ubiquitin E2s that can be leveraged by RING E3 ligases to drive specific 23 ubiquitination. 24 25 26 Keywords: DNA repair / E2 / Enzyme allostery / RING E3 / Ubiquitination 2 bioRxiv preprint doi: https://doi.org/10.1101/429076; this version posted September 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 27 Introduction 28 Ubiquitination is an essential, versatile and reversible post-translational modification system 29 that enables eukaryotic cells to reprogram the fate and function of the modified protein and 30 its connected pathway. The modification is accomplished by a sequential enzyme cascade 31 wherein ubiquitin’s C-terminus is first activated by an E1, is transferred onto the catalytic 32 cysteine of an E2 conjugase (E2~Ub) and finally E3 ligases mediate the covalent attachment 33 of ubiquitin onto a target lysine residue (Hochstrasser, 2009; Pickart, 2001). The Really 34 Interesting New Gene (RING) ligase proteins represent the largest E3 family (~600 members) 35 which share a zinc coordinating cross-brace motif termed RING domain (Freemont, 2000). 36 Mechanistically, while non-RING elements of E3 ligases specify the substrate, the RING 37 domains bind an E2 surface distal from the E2 active site and indirectly induce substrate 38 ubiquitination by stabilizing a productive E2~Ub conformation (Metzger et al., 2014). 39 Moreover, any of the seven surface lysine residues on ubiquitin or its N-terminus can be 40 targeted to build polyubiquitin chains, thus enabling diverse signals (Kulathu and Komander, 41 2012). Typically, RING-E2 interactions are found to be transient thus allowing E3s to switch 42 their E2 partners in order to assemble polyubiquitin signals on the substrate (Brown et al., 43 2014; Kelly et al., 2014; Rodrigo-Brenni and Morgan, 2007; Windheim et al., 2008). Around 44 35 ubiquitin E2s are found in mammals, several of which build chains (Stewart et al., 2016). 45 Mechanisms of chain-assembly are well understood and generally involve additional non- 46 covalent interactions between the E2 and the acceptor ubiquitin surface proximal to the 47 linkage site (Eddins et al., 2006; Liu et al., 2014; Middleton and Day, 2015; Petroski and 48 Deshaies, 2005; Rodrigo-Brenni et al., 2010; Wickliffe et al., 2011). In contrast however, far 49 less is known about how RING E3-E2 enzyme pairs attach a single ubiquitin directly on the 50 substrate surface in the case of monoubiquitination. 3 bioRxiv preprint doi: https://doi.org/10.1101/429076; this version posted September 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 51 Site-specific monoubiquitin signals feature prominently in fundamental DNA damage 52 response pathways (Al-Hakim et al., 2010; Uckelmann and Sixma, 2017). In eukaryotes, the 53 repair of toxic DNA interstrand cross-links (ICL) is mediated by the Fanconi Anemia (FA) 54 pathway, defects in which give rise to FA, a genome instability disorder typified by bone 55 marrow failure and high predisposition to cancers (Garaycoechea and Patel, 2014; Kottemann 56 and Smogorzewska, 2013). A key event in FA-ICL damage response is the site-specific 57 mono-ubiquitination of two large (~160kDa) structurally homologous proteins, FANCD2 58 (Garcia-Higuera et al., 2001) and FANCI (Sims et al., 2007; Smogorzewska et al., 2007) 59 (Lys561 and Lys523 respectively in humans), that signals the recruitment of repair factors 60 (Ceccaldi et al., 2016). The specific modification is mediated by the RING bearing protein 61 FANCL, present within a nine-protein FA core-complex E3 ligase (FANCA-FANCG- 62 FAAP20-FANCC-FANCE-FANCF-FANCB-FANCL-FAAP100) and the E2 Ube2T 63 (Machida et al., 2006; Meetei et al., 2003; Walden and Deans, 2014). FANCL’s central 64 region, a bi-lobed UBC (Ubiquitin conjugation fold)-RWD domain, facilitates direct 65 FANCD2/FANCI interaction (Cole et al., 2010; Hodson et al., 2011) while the C-terminal 66 RING domain selectively binds Ube2T over other E2’s (Hodson et al., 2014). Further, 67 genetic mutations in Ube2T, a Class III E2 with an unstructured C-terminal extension, have 68 recently been linked to a FA phenotype (Hira et al., 2015; Rickman et al., 2015; Virts et al., 69 2015). Accordingly, in in vitro assays, the isolated FANCL and Ube2T enzymes catalyse 70 FANCD2 monoubiquitination, although the modification levels are unexpectedly low (Alpi et 71 al., 2008; Hodson et al., 2014). Studies in frog egg extracts show majority of FANCD2 is 72 present in complex with FANCI (Sareen et al., 2012) while the cell based data indicate mono- 73 ubiquitination of either protein requires the presence of the partner (Alpi and Patel, 2009). 74 However, a crystal structure of the mouse FANCI-FANCD2 complex reveals an extended 75 heterodimer interface which buries the respective target lysine (Joo et al., 2011). Notably, the 4 bioRxiv preprint doi: https://doi.org/10.1101/429076; this version posted September 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 76 addition of structured or duplex DNA greatly stimulates FANCD2 ubiquitination and requires 77 the presence of FANCI (Longerich et al., 2014; Sato et al., 2012). The DNA binding 78 propensity of the FANCI-FANCD2 complex, absent in FANCL or Ube2T, is proposed to 79 induce local reconfiguration that could improve FANCL’s access to the target sites. Finally, 80 while FANCL alone induces low levels of ubiquitination, however when in a FANCB- 81 FANCL-FAAP100 sub-complex FANCD2 monoubiquitination levels improve by around 5- 82 fold. The added presence of a FANCC-FANCE-FANCF sub-complex progressively enhances 83 the tandem mono-ubiquitination of the FANCD2-FANCI complex (Rajendra et al., 2014; van 84 Twest et al., 2017). Thus, in the current model the presence of DNA, FANCI and additional 85 FA sub-complexes are all required for the modification however, underlying mechanisms for 86 the sub-complex induced enhancement are not well understood. First, FANCL dependent 87 FANCD2 ubiquitination has been observed in non-vertebrate species which lack an intact FA 88 core-complex, suggesting in part that the mechanism for the specific ubiquitination is 89 encoded within FANCL (Sugahara et al., 2012; Zhang et al., 2009). Second, global proteomic 90 profiling have uncovered several other lysine on human FANCD2 (22 sites) and FANCI (44 91 sites) that are ubiquitinated in vivo indicating the surface of both proteins are viable acceptors 92 (Kim et al., 2011; Udeshi et al., 2013). As ubiquitin signalling is influential in almost every 93 cellular process in eukaryotes, a major unanswered question is how specific signals are 94 assembled and regulated. The FA-ICL repair pathway is crucial for cellular homeostasis thus, 95 understanding how FANCL targets precise FANCD2 and FANCI sites for strict 96 monoubiquitination would provide valuable insights into the mechanics of this crucial DNA 97 damage response signal as well as how target and signal specificity is achieved in 98 ubiquitination. 99 In this study we show that FANCL activates Ube2T for ubiquitination through an allosteric 100 mechanism that is distinct from the typical RING E3 based catalysis.
Load more

Recommended publications
  • FANCL Sirna (H): Sc-45661
    SAN TA C RUZ BI OTEC HNOL OG Y, INC . FANCL siRNA (h): sc-45661 BACKGROUND STORAGE AND RESUSPENSION Defects in FANCL are a cause of Fanconi anemia. Fanconi anemia (FA) is an Store lyophilized siRNA duplex at -20° C with desiccant. Stable for at least autosomal recessive disorder characterized by bone marrow failure, birth one year from the date of shipment. Once resuspended, store at -20° C, defects and chromosomal instability. At the cellular level, FA is characterized avoid contact with RNAses and repeated freeze thaw cycles. by spontaneous chromosomal breakage and a unique hypersensitivity to DNA Resuspend lyophilized siRNA duplex in 330 µl of the RNAse-free water cross-linking agents. At least 8 complementation groups have been identified pro vided. Resuspension of the siRNA duplex in 330 µl of RNAse-free water and 6 FA genes (for subtypes A, C, D2, E, F and G) have been cloned. Phospho- makes a 10 µM solution in a 10 µM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM rylation of FANC (Fanconi anemia complementation group) proteins is thought EDTA buffered solution. to be important for the function of the FA pathway. FA proteins cooperate in a common pathway, culminating in the monoubiquitination of FANCD2 protein APPLICATIONS and colocalization of FANCD2 and BRCA1 proteins in nuclear foci. FANCL is a ligase protein mediating the ubiquitination of FANCD2, a key step in the FANCL shRNA (h) Lentiviral Particles is recommended for the inhibition of DNA damage pathway. FANCL may be required for proper primordial germ cell FANCL expression in human cells.
    [Show full text]
  • Further Insights Into the Regulation of the Fanconi Anemia FANCD2 Protein
    University of Rhode Island DigitalCommons@URI Open Access Dissertations 2015 Further Insights Into the Regulation of the Fanconi Anemia FANCD2 Protein Rebecca Anne Boisvert University of Rhode Island, [email protected] Follow this and additional works at: https://digitalcommons.uri.edu/oa_diss Recommended Citation Boisvert, Rebecca Anne, "Further Insights Into the Regulation of the Fanconi Anemia FANCD2 Protein" (2015). Open Access Dissertations. Paper 397. https://digitalcommons.uri.edu/oa_diss/397 This Dissertation is brought to you for free and open access by DigitalCommons@URI. It has been accepted for inclusion in Open Access Dissertations by an authorized administrator of DigitalCommons@URI. For more information, please contact [email protected]. FURTHER INSIGHTS INTO THE REGULATION OF THE FANCONI ANEMIA FANCD2 PROTEIN BY REBECCA ANNE BOISVERT A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN CELL AND MOLECULAR BIOLOGY UNIVERSITY OF RHODE ISLAND 2015 DOCTOR OF PHILOSOPHY DISSERTATION OF REBECCA ANNE BOISVERT APPROVED: Dissertation Committee: Major Professor Niall Howlett Paul Cohen Becky Sartini Nasser H. Zawia DEAN OF THE GRADUATE SCHOOL UNIVERSITY OF RHODE ISLAND 2015 ABSTRACT Fanconi anemia (FA) is a rare autosomal and X-linked recessive disorder, characterized by congenital abnormalities, pediatric bone marrow failure and cancer susceptibility. FA is caused by biallelic mutations in any one of 16 genes. The FA proteins function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). The monoubiquitination of FANCD2 and FANCI is a central step in the activation of the FA-BRCA pathway and is required for targeting these proteins to chromatin.
    [Show full text]
  • At Elevated Temperatures, Heat Shock Protein Genes Show Altered Ratios Of
    EXPERIMENTAL AND THERAPEUTIC MEDICINE 22: 900, 2021 At elevated temperatures, heat shock protein genes show altered ratios of different RNAs and expression of new RNAs, including several novel HSPB1 mRNAs encoding HSP27 protein isoforms XIA GAO1,2, KEYIN ZHANG1,2, HAIYAN ZHOU3, LUCAS ZELLMER4, CHENGFU YUAN5, HAI HUANG6 and DEZHONG JOSHUA LIAO2,6 1Department of Pathology, Guizhou Medical University Hospital; 2Key Lab of Endemic and Ethnic Diseases of The Ministry of Education of China in Guizhou Medical University; 3Clinical Research Center, Guizhou Medical University Hospital, Guiyang, Guizhou 550004, P.R. China; 4Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; 5Department of Biochemistry, China Three Gorges University, Yichang, Hubei 443002; 6Center for Clinical Laboratories, Guizhou Medical University Hospital, Guiyang, Guizhou 550004, P.R. China Received December 16, 2020; Accepted May 10, 2021 DOI: 10.3892/etm.2021.10332 Abstract. Heat shock proteins (HSP) serve as chaperones genes may engender multiple protein isoforms. These results to maintain the physiological conformation and function of collectively suggested that, besides increasing their expres‑ numerous cellular proteins when the ambient temperature is sion, certain HSP and associated genes also use alternative increased. To determine how accurate the general assumption transcription start sites to produce multiple RNA transcripts that HSP gene expression is increased in febrile situations is, and use alternative splicing of a transcript to produce multiple the RNA levels of the HSF1 (heat shock transcription factor 1) mature RNAs, as important mechanisms for responding to an gene and certain HSP genes were determined in three cell increased ambient temperature in vitro. lines cultured at 37˚C or 39˚C for three days.
    [Show full text]
  • The PI3K/Akt1 Pathway Enhances Steady-State Levels of FANCL
    This is a repository copy of The PI3K/Akt1 pathway enhances steady-state levels of FANCL. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/153365/ Version: Published Version Article: Dao, K.-H.T., Rotelli, M.D., Brown, B.R. et al. (6 more authors) (2013) The PI3K/Akt1 pathway enhances steady-state levels of FANCL. Molecular Biology of the Cell, 24 (16). pp. 2582-2592. https://doi.org/10.1091/mbc.E13-03-0144 Reuse This article is distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike (CC BY-NC-SA) licence. This licence allows you to remix, tweak, and build upon this work non-commercially, as long as you credit the authors and license your new creations under the identical terms. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ M BoC | ARTICLE The PI3K/Akt1 pathway enhances steady-state levels of FANCL Kim-Hien T. Daoa, Michael D. Rotellia, Brieanna R. Browna, Jane E. Yatesa,b, Juha Rantalac, Cristina Tognona, Jeffrey W. Tynera,d, Brian J. Drukera,e,*, and Grover C. Bagbya,b,* aKnight Cancer Institute, cDepartment of Biomedical Engineering, dDepartment of Cell and Developmental Biology, and eHoward Hughes Medical Institute, Oregon Health and Science University, Portland, OR 97239; bNorthwest VA Cancer Research Center, VA Medical Center Portland, Portland, OR 97239 ABSTRACT Fanconi anemia hematopoietic stem cells display poor self-renewal capacity Monitoring Editor when subjected to a variety of cellular stress.
    [Show full text]
  • Table S2.Up Or Down Regulated Genes in Tcof1 Knockdown Neuroblastoma N1E-115 Cells Involved in Differentbiological Process Anal
    Table S2.Up or down regulated genes in Tcof1 knockdown neuroblastoma N1E-115 cells involved in differentbiological process analysed by DAVID database Pop Pop Fold Term PValue Genes Bonferroni Benjamini FDR Hits Total Enrichment GO:0044257~cellular protein catabolic 2.77E-10 MKRN1, PPP2R5C, VPRBP, MYLIP, CDC16, ERLEC1, MKRN2, CUL3, 537 13588 1.944851 8.64E-07 8.64E-07 5.02E-07 process ISG15, ATG7, PSENEN, LOC100046898, CDCA3, ANAPC1, ANAPC2, ANAPC5, SOCS3, ENC1, SOCS4, ASB8, DCUN1D1, PSMA6, SIAH1A, TRIM32, RNF138, GM12396, RNF20, USP17L5, FBXO11, RAD23B, NEDD8, UBE2V2, RFFL, CDC GO:0051603~proteolysis involved in 4.52E-10 MKRN1, PPP2R5C, VPRBP, MYLIP, CDC16, ERLEC1, MKRN2, CUL3, 534 13588 1.93519 1.41E-06 7.04E-07 8.18E-07 cellular protein catabolic process ISG15, ATG7, PSENEN, LOC100046898, CDCA3, ANAPC1, ANAPC2, ANAPC5, SOCS3, ENC1, SOCS4, ASB8, DCUN1D1, PSMA6, SIAH1A, TRIM32, RNF138, GM12396, RNF20, USP17L5, FBXO11, RAD23B, NEDD8, UBE2V2, RFFL, CDC GO:0044265~cellular macromolecule 6.09E-10 MKRN1, PPP2R5C, VPRBP, MYLIP, CDC16, ERLEC1, MKRN2, CUL3, 609 13588 1.859332 1.90E-06 6.32E-07 1.10E-06 catabolic process ISG15, RBM8A, ATG7, LOC100046898, PSENEN, CDCA3, ANAPC1, ANAPC2, ANAPC5, SOCS3, ENC1, SOCS4, ASB8, DCUN1D1, PSMA6, SIAH1A, TRIM32, RNF138, GM12396, RNF20, XRN2, USP17L5, FBXO11, RAD23B, UBE2V2, NED GO:0030163~protein catabolic process 1.81E-09 MKRN1, PPP2R5C, VPRBP, MYLIP, CDC16, ERLEC1, MKRN2, CUL3, 556 13588 1.87839 5.64E-06 1.41E-06 3.27E-06 ISG15, ATG7, PSENEN, LOC100046898, CDCA3, ANAPC1, ANAPC2, ANAPC5, SOCS3, ENC1, SOCS4,
    [Show full text]
  • Western Blot Sandwich ELISA Immunohistochemistry
    $$ 250 - 150 - 100 - 75 - 50 - 37 - Western Blot 25 - 20 - 15 - 10 - 1.4 1.2 1 0.8 0.6 OD 450 0.4 Sandwich ELISA 0.2 0 0.01 0.1 1 10 100 1000 Recombinant Protein Concentration(mg/ml) Immunohistochemistry Immunofluorescence 1 2 3 250 - 150 - 100 - 75 - 50 - Immunoprecipitation 37 - 25 - 20 - 15 - 100 80 60 % of Max 40 Flow Cytometry 20 0 3 4 5 0 102 10 10 10 www.abnova.com June 2013 (Fourth Edition) 37 38 53 Cat. Num. Product Name Cat. Num. Product Name MAB5411 A1/A2 monoclonal antibody, clone Z2A MAB3882 Adenovirus type 6 monoclonal antibody, clone 143 MAB0794 A1BG monoclonal antibody, clone 54B12 H00000126-D01 ADH1C MaxPab rabbit polyclonal antibody (D01) H00000002-D01 A2M MaxPab rabbit polyclonal antibody (D01) H00000127-D01 ADH4 MaxPab rabbit polyclonal antibody (D01) MAB0759 A2M monoclonal antibody, clone 3D1 H00000131-D01 ADH7 MaxPab rabbit polyclonal antibody (D01) MAB0758 A2M monoclonal antibody, clone 9A3 PAB0005 ADIPOQ polyclonal antibody H00051166-D01 AADAT MaxPab rabbit polyclonal antibody (D01) PAB0006 Adipoq polyclonal antibody H00000016-D01 AARS MaxPab rabbit polyclonal antibody (D01) PAB5030 ADIPOQ polyclonal antibody MAB8772 ABCA1 monoclonal antibody, clone AB.H10 PAB5031 ADIPOQ polyclonal antibody MAB8291 ABCA1 monoclonal antibody, clone AB1.G6 PAB5069 Adipoq polyclonal antibody MAB3345 ABCB1 monoclonal antibody, clone MRK16 PAB5070 Adipoq polyclonal antibody MAB3389 ABCC1 monoclonal antibody, clone QCRL-2 PAB5124 Adipoq polyclonal antibody MAB5157 ABCC1 monoclonal antibody, clone QCRL-3 PAB9125 ADIPOQ polyclonal antibody
    [Show full text]
  • Human UBE2W Protein (His Tag)
    Human UBE2W Protein (His Tag) Catalog Number: 12725-H07E General Information SDS-PAGE: Gene Name Synonym: UBC-16; UBC16; 6130401J04Rik Protein Construction: A DNA sequence encoding the human UBE2W (Q96B02-12) (Met 1-Cys 151) was expressed, with a polyhistidine tag at the N-terminus. Source: Human Expression Host: E. coli QC Testing Purity: > 97 % as determined by SDS-PAGE Endotoxin: Protein Description Please contact us for more information. Ubiquitin-conjugating enzymes, also known as UBE2W, E2 enzymes and Stability: more rarely as ubiquitin-carrier enzymes, perform the second step of protein ubiquitination. The modification of protein with ubiquitin is an Samples are stable for up to twelve months from date of receipt at -70 ℃ important cellular mechanism for targeting abnormal or short-lived proteins for degradation. Ubiquitination involves at least three classes of enzymes: Predicted N terminal: Met ubiquitin-activating enzymes, or E1s, ubiquitin-conjugating enzymes, or Molecular Mass: E2s, and ubiquitin-protein ligases, or E3s. UBE2W is a member of the E2 ubiquitin-conjugating enzyme family. This enzyme is required for post- The recombinant human UBE2W comprises 166 amino acids and has a replicative DNA damage repair. It accepts ubiquitin from the E1 complex predicted molecular mass of 19.2 kDa.. It migrates as an approximately and catalyzes its covalent attachment to other proteins. It also catalyzes 18KDa band in SDS-PAGE under reducing conditions. monoubiquitination and "Lys-11"-linked polyubiquitination. UBE2W is also considered to regulate FANCD2 monoubiquitination. UBE2W exhibits Formulation: ubiquitin conjugating enzyme activity and catalyzes the monoubiquitination of PHD domain of Fanconi anemia complementation group L (FANCL).
    [Show full text]
  • Mosaic Origin of the Eukaryotic Kinetochore 2 Jolien J.E
    bioRxiv preprint doi: https://doi.org/10.1101/514885; this version posted January 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Mosaic origin of the eukaryotic kinetochore 2 Jolien J.E. van Hooff12*, Eelco Tromer123*#, Geert J.P.L. Kops2+ and Berend Snel1+# 3 4 1 Theoretical Biology and Bioinformatics, Biology, Science Faculty, Utrecht University, the Netherlands 5 2 Oncode Institute, Hubrecht Institute – KNAW (Royal Netherlands Academy of Arts and Sciences) and 6 University Medical Centre Utrecht, Utrecht, The Netherlands 7 3 Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom 8 9 * equal contribution as first author 10 + equal contribution as senior author 11 # corresponding authors ([email protected]; [email protected]) 1 bioRxiv preprint doi: https://doi.org/10.1101/514885; this version posted January 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 12 Abstract 13 The emergence of eukaryotes from ancient prokaryotic lineages was accompanied by a remarkable increase in 14 cellular complexity. While prokaryotes use simple systems to connect DNA to the segregation machinery during 15 cell division, eukaryotes use a highly complex protein assembly known as the kinetochore. Although conceptually 16 similar, prokaryotic segregation systems and eukaryotic kinetochore proteins share no homology, raising the 17 question of the origins of the latter.
    [Show full text]
  • The Effect of Increased Temperatures on the Antarctic Sponge Isodictya Sp
    bioRxiv preprint doi: https://doi.org/10.1101/416677; this version posted September 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Warm Temperatures, Cool Sponges: The Effect of Increased Temperatures on the Antarctic Sponge Isodictya sp. M. González-Aravena1*, N.J. Kenny2*^, M. Osorio1, A. Font1, A. Riesgo2, C.A. Cárdenas1^ 1 Departamento Científico, Instituto Antártico Chileno, Plaza Muñoz Gamero 1055, Punta Arenas, 6200965, Chile 2 Life Sciences Department, The Natural History Museum, Cromwell Road, London SW7 5BD, UK * Authors contributed equally to manuscript ^ Corresponding authors MGA: [email protected] NJK: [email protected] MO: [email protected] AF: [email protected] AR: [email protected] CAC: [email protected] Corresponding author details: NJK: +44 20 7942 6475 CAC: +56 61 2298124 1 bioRxiv preprint doi: https://doi.org/10.1101/416677; this version posted September 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract Although the cellular and molecular responses to exposure to relatively high temperatures (acute thermal stress or heat shock) have been studied previously, only sparse empirical evidence of how it affects cold-water species is available. As climate change becomes more pronounced in areas such as the Western Antarctic Peninsula, it has become crucial to understand the capacity of these species to respond to thermal stress.
    [Show full text]
  • Molecular Signatures Differentiate Immune States in Type 1 Diabetes Families
    Page 1 of 65 Diabetes Molecular signatures differentiate immune states in Type 1 diabetes families Yi-Guang Chen1, Susanne M. Cabrera1, Shuang Jia1, Mary L. Kaldunski1, Joanna Kramer1, Sami Cheong2, Rhonda Geoffrey1, Mark F. Roethle1, Jeffrey E. Woodliff3, Carla J. Greenbaum4, Xujing Wang5, and Martin J. Hessner1 1The Max McGee National Research Center for Juvenile Diabetes, Children's Research Institute of Children's Hospital of Wisconsin, and Department of Pediatrics at the Medical College of Wisconsin Milwaukee, WI 53226, USA. 2The Department of Mathematical Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA. 3Flow Cytometry & Cell Separation Facility, Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA. 4Diabetes Research Program, Benaroya Research Institute, Seattle, WA, 98101, USA. 5Systems Biology Center, the National Heart, Lung, and Blood Institute, the National Institutes of Health, Bethesda, MD 20824, USA. Corresponding author: Martin J. Hessner, Ph.D., The Department of Pediatrics, The Medical College of Wisconsin, Milwaukee, WI 53226, USA Tel: 011-1-414-955-4496; Fax: 011-1-414-955-6663; E-mail: [email protected]. Running title: Innate Inflammation in T1D Families Word count: 3999 Number of Tables: 1 Number of Figures: 7 1 For Peer Review Only Diabetes Publish Ahead of Print, published online April 23, 2014 Diabetes Page 2 of 65 ABSTRACT Mechanisms associated with Type 1 diabetes (T1D) development remain incompletely defined. Employing a sensitive array-based bioassay where patient plasma is used to induce transcriptional responses in healthy leukocytes, we previously reported disease-specific, partially IL-1 dependent, signatures associated with pre and recent onset (RO) T1D relative to unrelated healthy controls (uHC).
    [Show full text]
  • Supplementary Figures
    Supplementary Fig. S1 NOTCH1 NOTCH2 NOTCH3 NOTCH4 Co-mutation POPLAR/OAK (n=255) Rizvi/MSKCC (n=49) LUSC TCGA (n=469) Total (n=773) POPLAR/OAK (n=598) Rizvi/MSKCC (n=299) Non-LUSC TCGA (n=562) Total (n=1159) 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 Mutational rate (%) Mutational rate (%) Mutational rate (%) Mutational rate (%) Mutational rate (%) Supplementary Fig. S2 A POPLAR/OAK Univariable analysis Multivariable analysis PFS HR (95% CI) p value HR (95% CI) p value NOTCH1 (mut, WT) 0.70 (0.43-1.13) 0.140 0.69 (0.43-1.10) 0.121 NOTCH2 (mut, WT) 0.89 (0.52-1.53) 0.680 0.87 (0.51-1.48) 0.601 NOTCH3 (mut, WT) 0.74 (0.40-1.39) 0.354 0.73 (0.39-1.36) 0.320 NOTCH4 (mut, WT) 1.21 (0.74-1.98) 0.442 1.23 (0.75-2.00) 0.415 NOTCH1/2/3 (mut, WT) 0.72 (0.52-1.00) 0.051 0.25 0.5 1 2 4 0.25 0.5 1 2 4 Favors NOTCHmut Favors NOTCHWT Favors NOTCHmut Favors NOTCHWT B Rizvi Univariable analysis Multivariable analysis PFS HR (95% CI) p value HR (95% CI) p value NOTCH1 (mut, WT) 0.69 (0.34-1.41) 0.313 0.65 (0.32-1.34) 0.244 NOTCH2 (mut, WT) 0.47 (0.17-1.27) 0.136 0.41 (0.14-1.18) 0.098 NOTCH3 (mut, WT) 0.67 (0.30-1.51) 0.329 0.63 (0.28-1.42) 0.266 NOTCH4 (mut, WT) 0.92 (0.47-1.80) 0.799 1.23 (0.60-2.52) 0.570 NOTCH1/2/3 (mut, WT) 0.56 (0.34-0.92) 0.023 0.25 0.5 1 2 4 0.25 0.5 1 2 4 Favors NOTCHmut Favors NOTCHWT Favors NOTCHmut Favors NOTCHWT C POPLAR/OAK Univariable analysis Multivariable analysis OS HR (95% CI) p value HR (95% CI) p value NOTCH1 (mut, WT) 1.01 (0.60-1.71) 0.963 1.00 (0.59-1.68) 0.987 NOTCH2 (mut, WT) 0.94 (0.50-1.77)
    [Show full text]
  • Ubiquitinating Enzyme Systems Eric Yao, Msc
    TECHNICAL BULLETIN Ubiquitinating Enzyme Systems Eric Yao, MSc. Scientist, Product Management - SignalChem Biotech Inc. Target Ub Recognizing Domain Target A E1 E2 Ub Target P Ub P Ub P E3 Ub Ub Poly-Ub A P P E1 E2 E2 - Interacting Ub P Domain Ub Ub Ub Monomeric 26 S Proteosome Ubiquitin Polymeric Ubiquitin Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Deconjugation/ Ub Ub Ub Recycling Target Degradation Protein modifications by ubiquitin (Ub) or ubiquitin-like proteins (UBLs) participate in many critical cellular processes such as cell-cycle regulation, DNA repair, oncogenesis, antiviral pathways and most notably, proteasomal degradation of target proteins. Ubiquitination and modification by UBLs share a similar catalytic cascade which requires the sequential action of three classes of enzymes: E1 activating enzymes, E2 conjugating enzymes and E3 ligases. Recent research has linked dysregulation of the Ub/UBLs modification system to numerous diseases including cancer, immunological disorders and neurodegeneration. Thus, the high substrate specificity provided by combinations of over 30 E2’s and over 600 E3’s makes these enzymes emerging drug targets. In response to a growing market demand, SignalChem has developed an extensive array of products encompassing enzymes, Ub/UBL modifiers and substrates in the ubiquitination, SUMOylation, ISGylation and NEDDylation processes. Using Promega’s AMP-GloTM technology and an optimized assay protocol, we have identified and validated a variety of functional combinations of the enzyme components. With the established protocol, each enzyme in the catalytic cascade has been assessed for their activity towards generation of free AMP. In addition, inhibition profiles of the ubiquitinating enzymes have been obtained using the assay system, further demonstrating their potential to be used in high-throughput screening to identify lead compounds for drug discovery and development programs.
    [Show full text]